Figure 6

Semi-quantitative RT-PCR for (A) Igf-2 and (B) Gfp C2MG and C2MIG cells transfected with RNAi constructs respectively targeting Igf-2 (pShagIgf-2, Ii) and Gfp (pShagGfp, Gi). (C) Densitometry of Igf-2 signal expressed as a ratio of α-tubulin controls confirms that pShagIgf-2 (Ii) but not pShagGfp (Gi) abolishes the Igf-2 message (* statistically significant, p < 0.05, Student t-test). (A, C) Endogenous Igf-2 mRNA is detected at very low levels in undifferentiated (Un) wild-type (C2MG) and C2MIG cells and is upregulated following differentiation (Di). Substantial induction of ectopic Igf-2 mRNA is seen in C2MIG expressing cells following differentiation. pShagIgf-2 abolishes both endogenous and ectopic (C2MIG) Igf-2 mRNAs. (B, C) Gfp mRNA is not present in undifferentiated C2MG or C2MIG cells but is detected in both cell lines following differentiation. pShagGfp abolishes Gfp mRNA in these cells. (D) C2MIG cells transfected with pShagIgf-2 show a substantial reduction in the proportion of their fast-myosin positive myotubes which is statistically significant (Student t-test, p = 0.004, indicated by **) and, (D) a concomitant increase in the proportion of slow myosin positive myotubes (statistically significant, P = 0.07 *). Abolishing endogenous Igf-2 expression in C2MG cells results in, (D) a smaller but statistically significant (p = 0.02, **) drop in the number of fast-myosin positive myotubes but (D) does not increase the number of slow-myosin positive myotubes. Abolishing Gfp using pShagGfp has no effect on the proportion of (D) FMyHC or (D) SMyHC positive myotubes present in either C2MG or C2MIG cells. Igf-2 RNAi abolishes Igf-2 immunostaining in (F) C2MIG myotubes. (G) Igf-2 is strongly expressed in differentiated C2MIG myotubes (F-G; black arrows indicate myotubes). (G) Igf-2 is present in small numbers of mono-nuclear bipolar cells consistent with the reported expression of muscle Creatine Kinase (mCK) just prior to differentiation (white arrow). Total myotube number for cells treated with lipofectamine, pShagGfp and pShagIgf-2 was analysed by ANOVA and showed that the number of myotubes generated was not affected by the treatment regime, p = 0.64 (MG), p = 0.23 (MIG).